Nano-coupling agent for visualizing spatiotemporal precise control of no and preparation method and application thereof

Abstract

The application belongs to the field of biomedical materials, and particularly relates to a preparation method and application of a nano-coupling agent capable of visualizing spatiotemporal precise control of nitric oxide release. The nano-coupling agent comprises oxidized hyaluronic acid, aminated hyaluronic acid and a nitric oxide donor-loaded liposome; wherein the nitric oxide donor liposome is dispersed in a dynamic crosslinking network formed by the Schiff base reaction of the oxidized hyaluronic acid and the aminated hyaluronic acid. The nano-coupling agent can promote the transdermal delivery of nitric oxide under the action of ultrasound and achieve spatiotemporal controllable release and visualized monitoring.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a nano-coupler for the precise spatiotemporal release of NO through visualization, its preparation method, and its application. Background Technology

[0002] Nitric oxide is an important endogenous gaseous signaling molecule involved in various physiological and pathological processes, including vasodilation, immune regulation, cell migration, apoptosis, and transcriptional regulation. Due to its short half-life, rapid diffusion rate, and difficulty in controlling local concentrations, direct application of nitric oxide makes it challenging to establish stable, safe, and effective concentrations in lesion tissues.

[0003] Transdermal drug delivery is one of the ideal methods for treating skin diseases. Ultrasound, as a non-invasive mechanical wave, can facilitate drug crossing the skin barrier through cavitation, acoustic flow, and local micro-perturbations, and can also be used for tissue imaging, enhanced penetration delivery, and treatment process control. During ultrasound operation, the physical state of the coupling agent has a significant impact on the efficiency of ultrasound energy transfer. Traditional ultrasound coupling agents are mostly hydrogels or polymer systems, primarily functioning to eliminate air between the probe and the skin and improve the continuity of sound wave transmission; however, they typically lack drug loading, responsive release, and therapeutic functions.

[0004] For hydrogel-based ultrasound coupling systems, the polymer concentration, crosslinking density, and tissue adhesion of the material all affect its ultrasound coupling state. When the system concentration is too low or too high, the material is too fluid, making it difficult to form a stable coating layer on the skin surface, easily leading to loss, resulting in insufficient local retention and NO controlled release capacity. Furthermore, ultrasound can not only be used as a means to promote penetration and trigger release, but also as an imaging modality to observe the distribution and area of ​​action of the material in local tissues. Ultrasound imaging can monitor changes in acoustic signals at the drug delivery site. For nitric oxide delivery systems requiring precise spatiotemporal control, it not only enables a "visible" drug delivery process, but also allows for optimization of the ultrasound action site, duration, and intensity based on imaging feedback, thereby improving the spatial selectivity of NO release and therapeutic safety.

[0005] However, traditional ultrasound coupling agents are only used to improve sound wave transmission between the probe and the skin, and usually do not have ultrasound imaging, drug loading, or responsive controlled release functions. Although some existing drug delivery systems can promote transdermal absorption through ultrasound, they are difficult to simultaneously achieve visualization of the material delivery process and controllability of the NO release process. Therefore, it is necessary to construct a nano-coupling agent that combines ultrasound imaging, transdermal permeation enhancement, and spatiotemporal controlled NO release functions. Summary of the Invention

[0006] To address the shortcomings and deficiencies of existing technologies, this invention provides a nano-coupler for the precise spatiotemporal release of NO, its preparation method, and its application.

[0007] In a first aspect, the present invention provides a method for preparing a nano-coupling agent, comprising the following steps:

[0008] (1) Preparation of oxidized hyaluronic acid (OHA) and aminolated hyaluronic acid (HA-ADH):

[0009] Sodium hyaluronate and sodium periodate were dissolved in pure water and stirred in the dark to stop the oxidation reaction. Then, ethylene glycol was added to terminate the oxidation reaction. The resulting solution was then dialyzed in pure water and lyophilized to obtain oxidized hyaluronic acid, which was dissolved in PBS or pure water to obtain OHA solution.

[0010] Sodium hyaluronate was completely dissolved in pure water to obtain a sodium hyaluronate solution. 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were dissolved in an aqueous solution of DMSO and added dropwise to the sodium hyaluronate solution to activate the carboxyl groups. After stirring the reaction, adipic acid dihydrazide (ADH) was added to adjust the pH to 6.6-6.9. The resulting solution was dialyzed with pure water, lyophilized, and then dissolved in PBS or pure water to obtain a HA-ADH solution.

[0011] (2) Preparation of nitric oxide-loaded nano-coupling agents:

[0012] A liposome capable of releasing NO (i.e., nitric oxide nanocarrier NL) was obtained by preparing liposomes from phospholipids and ionic NO donor materials.

[0013] The NO-releasing liposomes were added to a HA-ADH solution and mixed thoroughly. Then, an OHA solution was added to allow HA-ADH and OHA to undergo in-situ cross-linking through a Schiff base dynamic reaction, forming a nano-coupling agent loaded with nitric oxide nanocarriers.

[0014] In some embodiments, the nitric oxide nanocarrier NL is obtained by reacting a cationic polymer with cholesterol chloroformate, then dissolving it with sodium methoxide, introducing an inert gas after dissolution, and then introducing NO gas to react, thus obtaining the ionic NO donor material.

[0015] In some embodiments, when preparing OHA, the molecular weight of the sodium hyaluronate is 400-800 kDa; or the mass ratio of sodium hyaluronate to sodium periodate is (2-3):1; or the mass-volume ratio of sodium hyaluronate to pure water is (1-1.5) g:100 mL; or the stirring time in the dark is 4-8 h; the mass-volume ratio of sodium hyaluronate to ethylene glycol is (1-1.2) g:500 μL; or the dialysis time is 2-3 days.

[0016] In some embodiments, when preparing HA-ADH, the molecular weight of the sodium hyaluronate is 400-800 kDa; or the mass-to-volume ratio of the sodium hyaluronate to pure water is (0.8-1) g: 100 mL; or the mass ratio of the sodium hyaluronate, HOBT and EDC is (1-1.2): 2: (1-1.2).

[0017] Alternatively, the DMSO in the aqueous solution has a volume ratio of (1-1.5):1 with pure water; the carboxyl activation time is 1-2 h.

[0018] Or the mass ratio of sodium hyaluronate to ADH is (1-1.5):2; and / or the dialysis time is 2-3 days.

[0019] In some embodiments, when the mass of the nitric oxide nanocarrier NL is 1-3 mg, the mass ratio of HA-ADH to OHA is 2-3:1.

[0020] In some embodiments, the mass concentration of OHA is 4%, the mass concentration of HA-ADH is 4%-6%, and the volume ratio of HA-ADH to OHA is 2:1.

[0021] Preferably, the solvent for HA-ADH and OHA is PBS or pure water.

[0022] The mass concentration of OHA is 4%; the mass concentration of HA-ADH is 4-6%; the volume ratio of HA-ADH to OHA is 2:1; and the concentration of NL is 0.03-0.1%.

[0023] In some embodiments, when the mass of the NO-releasing liposome is 1 to 3 mg, the amount of HA-ADH used is 80 to 120 mg.

[0024] Secondly, the present invention provides a nano-coupling agent obtained by the above preparation method.

[0025] Thirdly, this invention provides the application of the above-mentioned nano-coupler in the controlled release of NO.

[0026] Fourthly, this invention provides the application of the aforementioned nano-coupler in ultrasound imaging.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. This invention constructs a nano-coupling agent composed of NO donor material, liposome carrier and dynamic cross-linked network of hyaluronic acid, which achieves stable loading and local retention of NO donor.

[0029] 2. This invention obtains suitable concentrations and ratios of HA-ADH and OHA, and finds that their regulation of the performance of nano-couplers is a key factor. By adjusting the concentration and volume ratio of the two, the gelation state of the material and the NO release effect can be controlled.

[0030] 3. This invention enables the release of NO to be controllable in time and space through ultrasonic parameter screening and material state regulation, which helps to avoid potential cytotoxicity caused by excessive NO release, while improving the safety and effectiveness of treatment.

[0031] 4. The OHA and HA-ADH used in this invention are both derived from hyaluronic acid derivatives, and have good biocompatibility, degradability and tissue affinity, making them suitable for local skin drug delivery and ultrasound-assisted delivery scenarios. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 Synthesis and characterization of nitric oxide liposomes (NL) prepared in Example 1; (A) Schematic diagram of NL synthesis. (B) 1H NMR spectra of Cho and Cho-PEI; (C) UV spectra of Cho-PEI, Cho-PEI / NONOate after reaction with Griess reagent; (D) TEM image of NL; (E) Particle size distribution and PDI of NL; (F) NO cumulative release curve of NL in PBS (pH 7.4, 37°C).

[0034] Figure 2 Characterization of amino-modified hyaluronic acid and oxidized hyaluronic acid obtained in Example 2. (A) Synthetic route of amino-modified hyaluronic acid; (B) Synthetic route of oxidized hyaluronic acid; (C) Infrared spectrum of oxidized hyaluronic acid; (D) NMR spectrum of amino-modified hyaluronic acid.

[0035] Figure 3 Example 3 illustrates the effect of different concentrations of amino-modified hyaluronic acid on the performance of nano-couplers.

[0036] Figure 4The following are the construction and characterization of the nano-coupling agent obtained in Example 4: (A) Schematic diagram of Gel@NL construction; (B) Scanning electron microscopy image of Gel@NL; (C) Storage modulus (G′) and loss modulus (G″) of Gel@NL; (D) Stress scanning experiment of Gel@NL; (E) Injectability analysis of Gel@NL; (F) Self-healing performance analysis of Gel@NL; (G) Skin application performance analysis of Gel@NL; (H) NO release behavior analysis of Gel@NL.

[0037] Figure 5 To explore the ultrasonic safety parameters of the nano-coupling agent obtained in Example 4; (A) the effect of different ultrasonic intensities (2.0, 3.5 and 5.0 W / cm²) and irradiation times (5, 10 and 15 min) on pig skin temperature; (B) the tissue safety evaluation of mouse skin using H&E staining and Masson staining.

[0038] Figure 6 The analysis of NO transdermal controlled release from different concentrations of amino-modified hyaluronic acid obtained in Example 3.

[0039] Figure 7 The results show the quantitative analysis of NO transdermal permeability under different liposome loading conditions; (A) The quantitative analysis of NO concentration in the receiving chamber after continuous ultrasonic treatment for 30 min with Gel@Cho-PEI / NONOate at an ultrasonic power density of 3.5 W / cm²; (B) The quantitative analysis of NO concentration in the receiving chamber after ultrasonic treatment with different NL loadings with different NL loadings.

[0040] Figure 8 Ultrasonic imaging analysis of the nano-coupler obtained in Example 4; (A) Ultrasonic imaging image of mouse heart; (B) Ultrasonic imaging image of mouse bladder; (C) Ultrasonic imaging image of human extensor muscle region. Detailed Implementation

[0041] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0042] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual* (2013), edited by Green and Sambrook, or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0043] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0044] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] In the following examples, room temperature refers to 10~35°C.

[0047] The preparation of the NO-releasing liposomes (nitric oxide nanocarriers NL) of this invention is based on the method for preparing NO-releasing liposomes (NL) described in Chinese Patent CN115400084A:

[0048] The preparation method of the ionic NO donor material includes the following steps:

[0049] (1) The cationic polymer is dissolved in an organic solvent, cholesterol chloroformate and triethylamine catalyst are added, and the reaction is carried out at 0-4°C. Then the temperature is heated to 5-35°C and the reaction is continued. After the reaction is completed, the mixture is concentrated, and hydrochloric acid is added to the concentrate to redissolve it. The concentrate is washed, precipitated and dried to obtain cholesterol modified by the cationic polymer. The molar ratio of the cationic polymer to cholesterol chloroformate is 1:1. The cationic polymer is hyperbranched polyethyleneimine with a molecular weight of 600.

[0050] (2) The cholesterol modified by the cationic polymer obtained in step (1) is dissolved in an organic solvent, and then sodium methoxide is added to continue dissolving. After dissolution, an inert gas is introduced, and then NO gas is introduced to react, so as to obtain the ionic NO donor material.

[0051] The method for preparing the NO-releasing liposome includes the following steps: dissolving phospholipids and the ionic NO donor material in organic solvent C, then evaporating to remove the organic solvent to obtain a thin film, which is the NO-releasing liposome; the phospholipid is soybean lecithin, and the mass ratio of the phospholipid to the ionic NO donor material is 1:0.2.

[0052] The structural formula of the cholesterol modified by the cationic polymer is shown below:

[0053]

[0054] Ultrasound, as a mechanical wave, is non-invasive, economical, and convenient, enabling precise spatial and temporal control of drug delivery. The ultrasonic cavitation effect can non-invasively deliver drugs through the skin barrier to the dermis. However, at commonly used ultrasound frequencies, drugs alone cannot effectively propagate through the air, necessitating a coupling agent as a medium for sound wave transmission. Therefore, we attempted to construct a system with both good mechanical properties and controllable NO release capability. Based on biosafety-friendly hyaluronic acid, we prepared oxidized hyaluronic acid (OHA) and aminoated hyaluronic acid (HA-ADH) through chemical modification, and achieved in-situ cross-linking between the two via a Schiff base reaction.

[0055] In some embodiments, the method for screening the ultrasonic treatment parameters of the above-mentioned nano-coupler includes the following steps:

[0056] Preferably, the method for selecting the ultrasound treatment parameters includes: using ex vivo porcine skin as an in vitro skin model, and under the condition of a fixed ultrasound working frequency of 1 MHz, setting ultrasound intensities of 2.0 W / cm², 3.5 W / cm², and 5.0 W / cm², and ultrasound irradiation times of 5 min, 10 min, and 15 min respectively, to perform ultrasound treatment on the ex vivo porcine skin treated with nano-coupling agent; during the ultrasound treatment, using an infrared thermal imager to monitor the changes in skin surface temperature in the irradiated area in real time, and selecting the optimal ultrasound parameters based on the temperature rise, temperature spatial distribution, and tissue tolerance. Further, mouse skin is treated using the selected ultrasound parameters. After treatment, skin tissue from the treated area is collected, fixed, embedded, and sectioned, and then subjected to H&E staining and Masson's trichrome staining.

[0057] NO-releasing liposomes (NL) were prepared according to the preparation method described in Example 4 of Chinese Patent CN115400084A. The NL was analyzed by 1H NMR spectroscopy of Cho and Cho-PEI, UV spectroscopy of Cho-PEI and Cho-PEI / NONOate (referring to the preparation method in Example 1 of CN115400084A) after reaction with Griess reagent, TEM images, particle size distribution, and PDI of NL. (F) NO cumulative release curve of NL in PBS (pH 7.4, 37℃). The results indicate that the constructed NL system possesses clear NO loading characteristics, a stable liposome structure, and excellent sustained-release performance.

[0058] The prepared NO donor Cho-PEI / NONOate was characterized, such as... Figure 1 As shown in Figure B, Cho-PEI exhibited peaks of methylene groups in both the PEI backbone and branches at 2.5 ppm–3 ppm, a Cho allyl peak at 5.0 ppm–5.5 ppm, and peaks of Cho methyl and methylene groups at 0.5 ppm–0.9 ppm, thus confirming the successful synthesis of Cho-PEI. To verify whether NO was involved in the composition of the liposome complex, UV characterization was performed, as shown in Figure B. Figure 1 As shown in Figure C, the characteristic UV absorption peak of the NONOate group appears at 252 nm in NL, which further confirms the presence of the NONOate group. The above results fully demonstrate that NO has been successfully loaded. Figure 1 The NL shown in D is a spherical vesicle structure with no significant change in morphology. After drying, the size is stable below 200 nm, and the hydrated particle size of NL is around 200 nm, with uniform PDI distribution. Figure 1 E). Simultaneously, the release of NO was investigated under simulated human physiological conditions, and the results are as follows: Figure 1 As shown in Figure F, the NL system releases NO at a relatively rapid rate in the early stages, with a cumulative NO release of approximately 60% within 5 hours. Subsequently, the release rate decreases, entering a stable and slow release state. After 12 hours, the NO release reaches nearly 80%, and NO release essentially ceases after 24 hours. These results indicate that the constructed NL system possesses clear NO loading characteristics, a stable liposome structure, and superior sustained-release performance.

[0059] The prepared NO-releasing liposomes (NL), Cho-PEI / NONOate, were used in the following experiments.

[0060] In some embodiments, a method for preparing a nano-coupling agent is disclosed, comprising the following steps:

[0061] (1) Preparation of solutions of oxidized hyaluronic acid (OHA) and aminolated hyaluronic acid (HA-ADH):

[0062] (2) Preparation of nitric oxide-loaded nano-coupling agents:

[0063] Nitric oxide nanocarrier NL was added to HA-ADH solution and mixed evenly. Then, OHA solution was added to allow HA-ADH and OHA to undergo in-situ cross-linking through Schiff base dynamic reaction, forming a nano-coupling agent loaded with nitric oxide nanocarrier.

[0064] The concentrations and ratios of HA-ADH and OHA are key parameters for controlling the state of the nano-coupler. By adjusting the concentrations of HA-ADH and OHA, the viscosity, flowability, and mechanical strength of the nano-coupler can be controlled.

[0065] Furthermore, when the concentrations of HA-ADH and OHA are low or high, the cross-linking density of the system is insufficient, the nano-coupling agent has strong fluidity, making it difficult to form a stable covering layer on the skin surface, which can easily lead to material loss, unstable ultrasonic coupling, and difficulty in controlling the release of NO.

[0066] In some embodiments, during ultrasonic coupling, the operating frequency is fixed at 1 MHz and the intensity is 3.5 W / cm².

[0067] With ultrasonic control, 4% and 6% concentrations of HA-ADH form a coupling agent with OHA. After the ultrasound is started, it can promote NL penetration and release NO. After the ultrasound is stopped, the release stops.

[0068] Example 1

[0069] The preparation method of oxidized hyaluronic acid and aminolated hyaluronic acid includes the following steps:

[0070] Preparation of OHA: First, 1 g of HA (400–800 kDa, H909937, Maclean) and 450 mg of sodium periodate were dissolved in 90 mL of pure water. After stirring at 25°C in the dark for 4 hours, 500 μL of ethylene glycol was added to terminate the reaction. Then, the solution was dialyzed against pure water for 3 days, lyophilized to obtain OHA, and stored at -20°C for later use.

[0071] Preparation of HA-ADH: 0.8 g of hyaluronic acid (400–800 kDa, H909937, Maclean) was completely dissolved in 100 mL of pure water. 1.8 g of HOBT (1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide) and 0.8 g of EDC (1-hydroxybenzotriazole) were dissolved in a 1:1 mixture of DMSO and pure water and added dropwise to the HA solution to activate the carboxyl groups. After 1 hour, 1.8 g of adipic acid dihydrazide was added to adjust the pH to 6.8. The resulting solution was dialyzed against pure water for 3 days and then centrifuged at 7000 rpm for 15 minutes. The supernatant was then transferred to a 2 kDa molecular weight dialysis bag and dialyzed at room temperature for 3 days, changing the dialysate 3 times daily. After drying, it was frozen and stored at -20 °C for later use.

[0072] use 1 HNMR spectroscopy measures its structural changes, such as Figure 2 As shown in Figure D, b and c are the methylene peaks on the ADH aliphatic chain, proving that HA was successfully aminated. The d-position is the proton absorption peak of the OHA aldehyde group, proving that HA was successfully oxidized to produce an aldehyde group. Simultaneously, OHA was characterized using FTIR, as shown... Figure 2 As shown in C, OHA is at 1730 cm. -1The presence of a characteristic absorption peak of the C=O group further confirms the successful preparation of OHA.

[0073] Example 2

[0074] The effect of the concentration ratio of oxidized hyaluronic acid to aminated hyaluronic acid on the performance of nano-coupling agents:

[0075] We found that the concentration of HA-ADH affects the gelation state and ultrasonic coupling performance of the nano-coupler, and can control the viscosity, flowability and mechanical strength of the nano-coupler.

[0076] The nano-coupling agent Gel@NL was prepared by adding the nitric oxide nanocarrier NL to a pure aqueous solution of HA-ADH and mixing them evenly. Then, a pure aqueous solution of OHA was added to allow HA-ADH and OHA to undergo in-situ cross-linking through a Schiff base dynamic reaction, forming a nano-coupling agent loaded with the nitric oxide nanocarrier.

[0077] 40 mg of lyophilized OHA was dissolved in 1 mL of pure water (4% mass concentration, 600 rpm, stirring time 12 h). HA-ADH solutions with mass concentrations of 2%, 4%, 6%, and 8% were prepared, i.e., 40, 80, 120, and 160 mg of lyophilized HA-ADH were dissolved in 2 mL of pure water (600 rpm, stirring time 12 h). After dissolution, 1 mg of NL, 2 mL of HA-ADH solution, and 1 mL of OHA solution were mixed and stirred at 600 rpm for 5 min. In-situ crosslinking was then performed via Schiff base reaction. Coupling agents of different concentrations were prepared according to the above method, and the rheological properties of the samples were tested. The results are as follows: Figure 3 As shown, when the HA-ADH concentration is 4% and 6%, the storage modulus (G′) is always higher than the loss modulus (G″) and remains relatively stable with frequency, indicating that the system mainly exhibits elastic behavior and has good structural stability. When the HA-ADH concentration is too low or too high (2% and 8%), the storage modulus (G′) is always lower than the loss modulus (G″), indicating that the system has insufficient cross-linking, strong fluidity, and is not conducive to forming a uniform and continuous ultrasonic coupling interface.

[0078] Example 3

[0079] Based on Example 2, 1 mg of NL was mixed in a mixed solution of 1 mL HA-ADH (4%, m / v) and 2 mL OHA (4%, m / v) (volume ratio 2:1) and stirred (600 rpm for 5 min). The mixture was then crosslinked in situ via Schiff base reaction to form the nano-coupling agent Gel@NL, which meets the requirement for rapid gelation in practical applications. Figure 4 B). Figure 4 Scanning electron microscopy (SEM) results showed that after Gel@NL was freeze-dried for 3 days, it exhibited a uniform and continuous three-dimensional porous network structure under the SEM. This loose and interconnected microstructure is conducive to the diffusion and release of active molecules. Figure 4 Gel rheological analysis in C showed that, within the test frequency range, the storage modulus (G′) of Gel@NL was consistently higher than the loss modulus (G″) and remained relatively stable with frequency variation. Figure 4 In the D-gel stress scanning experiment, as the stress increased, G′ and G″ crossed, indicating that the network structure underwent reversible damage; Figure 4 In alternating high and low strain cycle tests of F, the mechanical properties of Gel@NL recovered rapidly, indicating its good self-healing ability. This characteristic may be closely related to the presence of dynamic reversible Schiff base bonds. Furthermore, Gel@NL exhibits significant shear-thinning behavior with increasing shear rate, and its viscosity decreases significantly with increasing shear rate, endowing it with good injectability. Figure 4 E). This Gel@NL can maintain its structural integrity after extrusion, bending, and deformation under external force, further demonstrating its feasibility for application in complex physiological environments. Figure 4 G). Figure 4 The H results also showed that Gel@NL can release a certain amount of NO in the early stage to meet the immediate regulatory needs of the local microenvironment, and then enters a relatively slow and continuous release stage, which is conducive to maintaining a stable NO concentration level.

[0080] Example 4

[0081] To achieve effective ultrasound response control while ensuring tissue safety, this invention first systematically optimized the ultrasound parameters. Given that pig skin is similar to human skin in terms of tissue structure, thickness, and acoustic properties, excised pig skin was selected as the in vitro model. Therefore, under a fixed operating frequency of 1 MHz, we evaluated the combined effects of different ultrasound intensities (2.0, 3.5, and 5.0 W / cm²) and irradiation times (5, 10, and 15 min). Infrared thermal imaging technology was used to monitor the irradiated area in real time to visually reflect the spatial distribution and dynamic changes of skin surface temperature during ultrasound treatment. Figure 5 Thermal imaging results from A showed that, within a 15-minute irradiation cycle, the skin surface temperature at intensities of 2.0 and 3.5 W / cm² did not exceed the safe threshold of 40°C. In contrast, an ultrasound intensity of 5.0 W / cm² might introduce a non-negligible risk of thermal effects. Considering the ultrasound penetration efficiency, 3.5 W / cm² was selected as the optimal ultrasound intensity for subsequent in vivo experiments.

[0082] Based on this, we applied the selected ultrasound parameters (3.5 W / cm²) to mouse skin in vivo experiments and conducted a systematic pathological evaluation of the treated skin tissue.

[0083] Figure 5 H&E staining results of B showed that after ultrasound treatment, the epidermis and dermis of mouse skin remained intact, cell morphology was clear, and no obvious tissue necrosis or abnormal inflammatory infiltration was observed. Masson's trichrome staining further confirmed that collagen fibers were regularly arranged without abnormal breakage or excessively dense deposition. These results indicate that ultrasound treatment at 3.5 W / cm² for 15 min did not cause significant structural damage to skin tissue and demonstrated good tissue compatibility and biosafety.

[0084] Example 5

[0085] To further utilize ultrasound as an external physical trigger, the controlled release effect of different concentrations of coupling agent on NO in Example 2 was systematically evaluated. This study used a Franz diffusion cell device to construct a simulated skin permeation model. Ex vivo mouse whole skin was used as the permeation barrier, trimmed to an appropriate size, and fixed between the supply and receiving chambers. The receiving chamber was pre-filled with phosphate buffer (pH 7.4) to simulate the human physiological environment. A quantitative amount of Gel@NL was evenly applied to the stratum corneum surface of the skin, and ultrasound treatment was performed at a fixed frequency of 1 MHz and an intensity of 3.5 W / cm². Dynamic sampling was performed at a series of consecutive time points (5, 10, 15, 20, 25, 30, 35, 40, 45, 50 min). Each time 100 μL of permeate sample was drawn from the receiving chamber, an equal volume of PBS buffer was immediately replenished to maintain constant leakage conditions and volume. The collected samples were then mixed with Griess reagent and reacted for 15 min under light-protected conditions. The absorbance of the reaction solution at a wavelength of 540 nm was measured using a UV-Vis spectrophotometer. Figure 6 The results showed that ultrasound treatment did not lead to an instantaneous burst release of NO from the 4% and 6% concentration nano-couplers, but rather exhibited a controlled and enhanced release kinetics. The 2% and 8% concentrations of nano-couplers did not show any NO release characteristics. Ultrasound utilizes the acoustic cavitation effect to induce temporary relaxation of the stratum corneum structure and enhance tissue microfluidics, thereby synergistically improving transdermal penetration. The 4% and 6% concentrations of nano-couplers not only help improve the effective accumulation of NO in target tissues but also avoid the potential adverse effects of high-concentration NO accumulation in localized areas. Therefore, ultrasound-assisted nano-couplers achieve dual regulation of NO transdermal delivery efficiency and release behavior.

[0086] In the following embodiments, the fixed operating frequency of the ultrasound is 1 MHz and the intensity is 3.5 W / cm².

[0087] Example 6

[0088] The results of Example 5 showed that ultrasonic treatment did not induce a burst release of NO from the 4% and 6% concentration nano-couplers, but rather exhibited enhanced ultrasonic responsiveness and relatively controllable release kinetics. To further clarify the mediating role of the liposome carrier in the transdermal delivery of NO, 1 mg of NO donor Cho-PEI / NONOate was loaded into a mixture of HA-ADH solution (4%, m / v) and OHA solution (4%, m / v) at a volume ratio of 2:1. The mixture was in situ cross-linked via a Schiff base reaction, forming Gel@Cho-PEI / NONOate within approximately 5 min. In vitro transdermal permeability quantification analysis of Gel@Cho-PEI / NONOate was performed according to the method described in Example 5. Figure 7 As shown in Figure A, no NO was detected in the receiving chamber after 30 min of continuous ultrasound treatment. This result indicates that, in the absence of liposome carriers, free Cho-PEI / NONOate is difficult to effectively penetrate the skin barrier under the aforementioned ultrasound conditions. Combined with the transdermal results of the Gel@NL system, this demonstrates that after being loaded with liposomes, the NO donor can be delivered across the skin barrier under ultrasound by relying on the deformation ability of liposomes, thus confirming that liposome carriers play an important mediating role in the transdermal penetration of NO.

[0089] To further investigate the effect of NL loading on NO transdermal permeability, 1.5 mg and 3 mg of NL were loaded into a mixture of HA-ADH solution (4%, m / v) and OHA solution (4%, m / v) at a volume ratio of 2:1. The mixture was in-situ cross-linked via a Schiff base reaction, forming Gel@NL samples with different NL loadings within approximately 5 minutes. In vitro transdermal permeability quantitative analysis of each Gel@NL sample was performed according to the method described in Example 5. Figure 7 As shown in Figure B, the NO content detected in the receiving chamber did not significantly increase with increasing NL loading from 1.5 mg to 3 mg. These results indicate that, under ultrasound power density of 3.5 W / cm², increasing NL loading does not further enhance NO transdermal permeability. This differs from conventional studies that suggest NO transdermal concentration may gradually increase with increasing NL mass under the same ultrasound conditions. This suggests that the effective deformation and transdermal transport capacity of liposomes under these ultrasound conditions may have reached relative saturation, and NO transdermal permeability is limited by skin barrier flux or ultrasound-driven capacity.

[0090] Example 7

[0091] This invention systematically evaluated the ultrasound imaging performance of the material of Example 3 in different tissue environments. In mouse experiments, after coating it on the surface of the heart, high-frequency ultrasound imaging could clearly display the heart contour and its pulsating boundary. The echo signal was continuous and stable, and did not interfere with the identification of the normal dynamic structure of the heart, indicating that it has good acoustic coupling performance on the surface of dynamic organs. Figure 8 A). Simultaneously, in the bladder, a typical static fluid-containing organ model, the acoustic impedance matching between the bladder wall and the intracavitary fluid was significantly enhanced, resulting in clearer organ boundaries and significantly improved contrast of internal structures. Figure 8 B). Further imaging results of the superficial extensor muscle region of the human body showed that it effectively reduced surface artifacts and enhanced the echo signals of the muscle layer and deep tissues, with imaging clarity comparable to commercially available medical ultrasound coupling agents. Figure 8 C). This material can not only serve as a NO delivery carrier in ultrasound-triggered treatment, but also possesses excellent ultrasound imaging capabilities within the same system. This provides a visual basis for real-time positioning, status monitoring, and parameter adjustment during subsequent ultrasound treatment, facilitating precise control of the treatment process.

[0092] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

[0093] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a nano-coupling agent, characterized in that, The preparation method includes the following steps: (1) Preparation of oxidized hyaluronic acid and aminolated hyaluronic acid: Sodium hyaluronate and sodium periodate were dissolved in pure water and stirred in the dark to stop the oxidation reaction. Then, ethylene glycol was added to terminate the oxidation reaction. The resulting solution was then dialyzed in pure water and lyophilized to obtain oxidized hyaluronic acid, which was dissolved in PBS or pure water to obtain OHA solution. Sodium hyaluronate was completely dissolved in pure water to obtain a sodium hyaluronate solution. 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were dissolved in an aqueous solution of DMSO and added dropwise to the sodium hyaluronate solution to activate the carboxyl groups. After stirring the reaction, adipic acid dihydrazide (ADH) was added to adjust the pH to 6.6-6.

9. The resulting solution was dialyzed with pure water, lyophilized, and then dissolved in PBS or pure water to obtain a HA-ADH solution. (2) Preparation of nitric oxide-loaded nano-coupling agents: Liposomes capable of releasing NO were obtained by preparing phospholipids and ionic NO donor materials. The NO-releasing liposomes were added to a HA-ADH solution and mixed thoroughly. Then, an OHA solution was added to allow HA-ADH and OHA to undergo in-situ cross-linking through a Schiff base dynamic reaction, forming a nano-coupling agent loaded with nitric oxide nanocarriers.

2. The method for preparing the nano-coupling agent according to claim 1, characterized in that, The ionic NO donor material is obtained by reacting a cationic polymer with cholesterol chloroformate, then dissolving it in sodium methoxide, introducing an inert gas, and then introducing NO gas to react. Preferably, the method for preparing the NO-releasing liposomes includes: dissolving phospholipids and the ionic NO donor material in an organic solvent, then evaporating the organic solvent to obtain a film, which is the NO-releasing liposome; the phospholipid is soybean lecithin, and the mass ratio of the phospholipid to the ionic NO donor material is 1:0.

2.

3. The method for preparing the nano-coupling agent according to claim 1, characterized in that, When preparing OHA, at least one set of parameters is selected from the following AF groups: A. The molecular weight of the sodium hyaluronate is 400–800 kDa; B. The mass ratio of sodium hyaluronate to sodium periodate is (2-3):1; C. The mass-to-volume ratio of sodium hyaluronate to pure water is (1-1.5) g: 100 mL; D. The stirring time in the dark is 4–8 h; E. The mass-to-volume ratio of sodium hyaluronate to ethylene glycol is (1-1.2) g: 500 μL; F. The dialysis time is 2 to 3 days.

4. The method for preparing the nano-coupling agent according to claim 1, characterized in that, When preparing HA-ADH, at least one set of parameters is selected from the following AD groups: The molecular weight of sodium hyaluronate described in A is 400–800 kDa; The mass-to-volume ratio of sodium hyaluronate to pure water, as described in B, is (0.8–1) g : 100 mL. The mass ratio of sodium hyaluronate, HOBT, and EDC described in C is (1-1.2):2:(1-1.2). The DMSO in the aqueous solution is in a volume ratio of (1-1.5):1 with pure water; the carboxyl activation time is 1-2 hours. The mass ratio of sodium hyaluronate to ADH is (1-1.5):2; and / or the dialysis time is 2-3 days.

5. The method for preparing the nano-coupling agent according to any one of claims 1-4, characterized in that, When the mass of the liposome that can release NO is 1-3 mg, the mass ratio of HA-ADH to OHA is 2-3:

1.

6. The method for preparing the nano-coupling agent according to claim 5, characterized in that, When the mass concentration of OHA is 4%, and the mass concentration of HA-ADH is 4%-6%, the volume ratio of HA-ADH to OHA is 2:

1.

7. The method for preparing the nano-coupling agent according to claim 1, characterized in that, The solvent for HA-ADH and OHA is PBS or pure water; preferably, when the mass of the liposome that can release NO is 1 mg to 3 mg, the amount of HA-ADH used is 80 mg to 120 mg.

8. The nano-coupling agent obtained by any of the preparation methods described in claims 1-7.

9. The application of the nano-coupling agent according to claim 8 in controlled release of NO.

10. The application of the nanocoupler of claim 8 in ultrasound imaging.

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